A new method to prepare the vitamin A derivative mannosylretinyl phosphate (MRP) free from any dolichylmannosylphosphate (DMP) has been worked out. Using this method, it was found that cultured hepatocytes as well as their microsomal membranes synthesize both mannolipids in the ratio of 1 (MRP) to 3 (DMP). An investigation of the different role of MRP and DMP as intermediates in the biosynthesis of glycoproteins was conducted using (14C)MRP and (3H)DMP labeled in the mannosyl moiety. SDS-PAGE analysis of the acceptors, after removal of the lipids, showed that the two mannolipids served as donors of mannose to two distinct populations of glycoproteins. The mannosyl residues transferred from MRP were cleaved by treatment of the acceptors with alpha-mannosidase, demonstrating that an anomeric inversion from the beta-mannose of mannosylretinylphosphate to alpha-mannosyl residues of glycoproteins had occurred during transfer. Retinoid-treated 3T12 cells displayed increased adhesive properties. Metabolic labeling showed a markedly increased incorporation of (2-3H)mannose into a specific glycoprotein of the cell surface of 180 kilodaltons (gp180).